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| Status |
Public on Aug 10, 2020 |
| Title |
Multiplexed measurement of variant abundance and activity reveals VKOR topology, active site and human variant impact |
| Organisms |
Homo sapiens; synthetic construct |
| Experiment type |
Other
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| Summary |
Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin. Despite VKOR’s pivotal role in coagulation, its structure and active site remain poorly understood. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells.
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| Overall design |
Barcoded single amino acid variant libraries of VKOR variants were recombined into HEK293 and HEK293T cells previously engineered to contain a Bxb1 recombination site at the AAVS1 locus. Upon sorting cells for APC or eGFP expression, genomic DNA was extracted. Barcodes were amplified and counted with Illumina sequencing. Barcodes were associated back to the corresponding VKOR by comparison with a barcode-variant map previously created by sequencing the variant library plasmids using Illumina sequencing.
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| Contributor(s) |
Fowler DM |
| Citation(s) |
32870157 |
| Submission date |
May 05, 2020 |
| Last update date |
Sep 08, 2020 |
| Contact name |
Douglas Fowler |
| Organization name |
University of Washington
|
| Department |
Genome Sciences
|
| Lab |
Fowler
|
| Street address |
3720 15th Ave NE
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platforms (2) |
| GPL17769 |
Illumina MiSeq (synthetic construct) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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| Samples (14)
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| Relations |
| BioProject |
PRJNA630502 |
| SRA |
SRP260123 |
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